Ceramide-1-phosphate transfer protein enhances lipid transport by disrupting hydrophobic lipid–membrane contacts

Cellular distributions of the sphingolipid ceramide-1-phosphate (C1P) impact essential biological processes. C1P levels are spatiotemporally regulated by ceramide-1-phosphate transfer protein (CPTP), which efficiently shuttles C1P between organelle membranes. Yet, how CPTP rapidly extracts and inserts C1P into a membrane remains unknown. Here, we devise a multiscale simulation approach to elucidate biophysical details of CPTP-mediated C1P transport. We find that CPTP binds a membrane poised to extract and insert C1P and that membrane binding promotes conformational changes in CPTP that facilitate C1P uptake and release. By significantly disrupting a lipid’s local hydrophobic environment in the membrane, CPTP lowers the activation free energy barrier for passive C1P desorption and enhances C1P extraction from the membrane. Upon uptake of C1P, further conformational changes may aid membrane unbinding in a manner reminiscent of the electrostatic switching mechanism used by other lipid transfer proteins. Insertion of C1P into an acceptor membrane, eased by a decrease in membrane order by CPTP, restarts the transfer cycle. Most notably, we provide molecular evidence for CPTP’s ability to catalyze C1P extraction by breaking hydrophobic C1P–membrane contacts with compensatory hydrophobic lipid–protein contacts. Our work, thus, provides biophysical insights into how CPTP efficiently traffics C1P between membranes to maintain sphingolipid homeostasis and, additionally, presents a simulation method aptly suited for uncovering the catalytic mechanisms of other lipid transfer proteins.

di erences described by PCs 1-4 and the distributions of PC values sampled in solution-phase and membranebound simulations of both apo and C1P-bound forms of CPTP: S1 PC1 describes a concerted rotation about helix -6, which approximates the surface of the membrane. Negative values of PC1 correspond to structures in which helix -2 is located closer and more parallel to the membrane surface, whereas positive values correspond to structures with helix -2 orientated more perpendicular to the membrane surface. We find that structures of the C1P-bound form of CPTP bound to the membrane have increased values of PC1 on average compared to either the C1P-bound form in solution or the apo form. Thus, membrane binding of the C1P-bound form promotes a concerted reorientation that aids the opening of gating helix -2 and that positions a widened entrance to CPTP's hydrophobic cavity at the membrane surface.
PC2 describes an internal reorganization of CPTP's helices that rotates the sides of its sandwich-like structure relative to each other (as if two stacked planar sheets were rotated relative to each other). Such changes captured by PC2 are evocative of a cleft-like gating mechanism (Fig 1 and S1 Fig) [16]. Of the first four PCs, variation along PC2 best captures di erences between the apo and C1P-bound forms of CPTP, regardless of if CPTP is in solution or bound to the membrane. Indeed, C1P uptake (or release) results in substantial rearrangement of the sides of CPTP's sandwich-like structure relative to each other (Fig 1  and S1 Fig). Membrane-bound structures of both the apo and C1P-bound forms of CPTP have increased values of PC2 on average compared to their respective solution-phase structures. Thus, membrane binding promotes a consistent change in the cleft to CPTP's hydrophobic cavity.
PC3 describes a concerted rotation orthogonal to that of PC1; if motion along PC1 were described as 'rocking side-to-side', then motion along PC3 would be described as 'rocking forward-and-backward'. Conformational ensembles of the apo and C1P-bound forms sampled in both solution-phase and membranebound simulations exhibit similar variation along PC3.
PC4 describes an internal reorganization of CPTP's helices di erent from that of PC2. While solutionphase structures of CPTP have similar average values of PC4, the membrane-bound structures of the apo and C1P-bound forms have average values di erent from each other and from the solution-phase structures.
Overall, PCA indicates that structures of the apo and C1P-bound forms di er both in solution and bound to the membrane, and that membrane binding can promote opening of gating helix -2 and conformational changes suggestive of a cleft-like gating mechanism. S2 Note B: Definition of Q. The fraction of contacts C1P makes with CPTP when fully inside its hydrophobic cavity, Q, was used as a second order parameter (or collective variable) for biased simulations. Q was chosen to reliably identify configurations with C1P inside CPTP versus configurations with C1P outside CPTP and to enhance the sampling of CPTP-C1P interactions. Since r LxS only describes C1Pmembrane interactions, it accomplishes neither of these things, while Q does. Specifically, CPTP-C1P contact pairs used to calculate Q were selected to capture: 1. Hydrophobic contacts between carbons of C1P and carbons of residues lining CPTP's hydrophobic cavity. Carbon-carbon (CC) pairs were selected based on their average distance, d C1P≠CPTP , in solution-phase simulations of the C1P-bound form of CPTP (Fig B panel A). To minimize the computational expensive of calculating Q during biased simulations, which increases with the number of CC pairs considered, while accurately identifying configurations with C1P inside CPTP's hydrophobic cavity, a cuto of d C1P≠CPTP AE 7.8Å was used to select these CC pairs. Residues with these CC pairs used to calculate Q are shown in Fig B panel C. 2. Polar contacts between C1P's headgroup and sphingoid backbone and residues at the entrance to CPTP's hydrophobic cavity. Heavy atom pairs were selected based on d C1P≠CPTP (Fig B panel B).
To minimize the computational expense of calculating Q during biased simulations while capturing all important polar contacts, a cuto of d C1P≠CPTP AE 5.5Å was used to select these pairs. Residues with these polar atom pairs used to calculate Q are shown in Fig   Based on these criteria for selecting contact pairs, 1,176 atom pairs are used to calculate Q using Eq. 4 given in the Methods. We confirmed that Q reliably identifies configurations with C1P fully inside CPTP's hydrophobic cavity and orientated as in crystal structures from configurations with C1P outside, partially inside, or improperly orientated within CPTP's cavity. To do so, we monitored the value of Q during all-atom simulations in which C1P enters into CPTP's hydrophobic cavity from the solvent. While the relaxation process that occurs during these simulations is not cellularly relevant, it is su ciently rapid to observe in unbiased simulations. Thus, we are able to harvest multiple all-atom trajectories of C1P entry from solvent and use them to benchmark Q. Five simulations, each initialized with C1P randomly placed in the solvent around the apo form of CPTP, were performed using the same parameters as the all-atom solution-phase simulations described in the Methods. Each simulation was run for a maximum of 2 µs or until both tails of C1P were inserted into CPTP and no longer exposed to solvent based on visual inspection. Fig C shows the value of Q during each of these simulations and the final configuration of C1P bound to CPTP. In three of the simulations (shown in green, cyan, and blue in Fig C) created by helices -N and -4. These trajectories are not representative of C1P uptake from a membrane since insertion through helices -N and -4, which are fully exposed to solvent when CPTP is bound to a membrane (Fig 2), would require C1P to become fully solvated before entering CPTP's hydrophobic cavity. Thus, they serve as valuable tests of using Q to accurately identify configurations with C1P properly housed in CPTP's cavity. Q = 1 when C1P is properly housed inside CPTP's cavity, whereas Q never surpasses 0.5 in these trajectories. In the other two trajectories (shown in magenta and red in Fig C), C1P inserts through the entrance to CPTP's hydrophobic cavity as occurs when it's extracted from a membrane. In both trajectories, C1P's tails enter individually. In the trajectory shown in magenta in Fig C, the second tail fails to enter within 2 µs. In the trajectory shown in red in Fig C, both tails enter within 700 ns. In both trajectories, Q rapidly changes from Q ¥ 0.1 to Q ¥ 0.6 when the first tail enters, and, in the red trajectory, then changes rapidly again when the second tail enters. Thus, Q distinguishes di erent ways that C1P can bind to CPTP and can be used to enhance the sampling of interactions between C1P and CPTP. We note that these trajectories do not provide any evidence that Q is the reaction coordinate [32-34] (or necessarily a component of the reaction coordinate) for CPTP-mediated C1P transport.